Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by Loop-Mediated Isothermal Amplification (LAMP)

<p><b>Background:</b> The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.</p> <p><b>Methodology/Principal Findings:</b> For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.</p> <p><b>Conclusions/Significance:</b> This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.</p&gt

Socially sensitive regulation for water services

The provision of essential services such as water and sanitation may be considered a first step towards social inclusion. The overall sustainability of water and sanitation services also depends on social considerations. This paper explores the relationship between the regulator and the utility in the context of service provision for low-income users. It presents a general background to regulation in the water sector, along with some of the challenges faced by governments and regulators when implementing private sector involvement. Drawing upon the authors’ experience of water services management including regulation and private sector participation (PSP) in the water sector, the paper is based on a review of the literature, discussion with relevant professionals and an examination of a number of projects. The authors detail the role of the regulator and identify recurring themes relating to regulation and the poor. The shortcomings of specific projects are highlighted not as criticisms, but in the interest of sharing of knowledge and improving services to the poor in the long run. The paper includes suggestions on how regulation of water services could be undertaken in a low-income environment. The authors conclude that if water utilities are to perform in a socially sensitive manner, appropriate regulatory regimes are necessary

Disorders of sexual differentiation as seen at Kenyatta National Hospital

Background: Disorders of sexual differentiation (DSD) are a group of congenital anomalies characterised by discordance between genetic, gonadal and phenotypic sex. There has been remarkable evolution in management over the last decade, including nomenclature, diagnosis and management. There has also been increased awareness and interest from patients and the public, including legal opinion. There has however been no local study to document and evaluate management in Kenya.Objective: To describe management of patients presenting with DSD at Kenyatta National Hospital (KNH) over a 10 year period.Design: Retrospective descriptive study.Setting: Kenyatta National Hospital.Subjects: Patients diagnosed with DSD.Results: A total of 30 patients whose charts were available were reviewed. Age ranged from birth to 19 years (median 5 months) at diagnosis. Presumed gender was assigned at birth in 28 patients. Karyotyping was available in 23(76%) patients. Other common tests included blood tests (23, 76%), ultrasound scan (14, 47%), contrast studies (3, 10%) and exploratory laparoscopy or laparotomy. 46XX and 46XY DSD were the most common conditions encountered (13, 43% and 7, 23% respectively). The commonest operations were correction of hypospadias and orchidopexy (55%), followed by feminising genitoplasty (16%). Only two patients had gender reassignment. Fifteen patients were asked how they feel about their current gender, and 11 expressed satisfaction.Conclusion: DSD is a relatively rare condition. There are also no strict protocols being followed. Management at KNH is acceptable although there is a lack of facilities to carry out many requisite investigations

Site characterization and systems analysis in Central Mekong

The systems addressed in this chapter and in the CGIAR Research Program on Integrated Systems for the Humid Tropics (Humidtropics) broadly include natural systems comprising biophysical, resource and climate realities; social systems made up of people, societies and their institutions; and, what some term as artificial systems built on elements of the first two (Checkland 1981). Agricultural systems, for example, modify natural systems for productive use, add infrastructure to provide markets, and modify human institutions to organize labour and services to enable the agricultural system to function. Regardless of how systems are categorized, they can be simplistically deconstructed into components and the interactions between them. In this chapter we characterize some of the Central Mekong systems, and also address some of the system dynamics, at two basic levels of resolution. Section 2 addresses regional agricultural systems consisting of one or more districts within a country, and includes variations in natural and social systems in addition to agricultural systems. Five regional cases that reflect the diversity across the Central Mekong Action Area are examined and compared. The authors focus on systems at the community or local landscape level, particularly the individual farm household component, and the variation between households within the landscape. Variables include household agricultural practices, household resources, capacity, and links to markets and institutions. Section 3 looks at diversity in the variables among farm households and the implications for livelihoods and well-being. Section 4 examines food security levels arising from specific farm household strategies and performance, how the two are related, and the implications for potential farm interventions. We conclude by comparing the types of systems examined, the differences in types of tools needed, and the differences in questions asked and learning generated. Throughout this chapter, authors refer to data from reports and articles that interested readers can find in Annex I

Comparison of pap smear, visual inspection with acetic acid, human papillomavirus DNA-PCR testing and cervicography

Objective: To assess the test qualities of four screening methods to detect cervical intra-epithelial neoplasia in an urban African setting. Method: Six hundred fiftythree women, attending a family planning clinic in Nairobi (Kenya), underwent four concurrent screening methods: pap smear, visual inspection with acetic acid (VIA), PCR for high risk human papillomavirus (HR HPV) and cervicography. The presence of cervical intra-epithelial neoplasia (CIN) was verified by colposcopy or biopsy. Result: Sensitivity (for CIN2 or higher) and specificity (to exclude any CIN or cancer) were 83.3% (95% CI [73.6, 93.0]) and 94.6% (95% CI [92.6, 96.5]), respectively, for pap smear; 73.3% (95% CI [61.8, 84.9]) and 80.0% (95% CI [76.6, 83.4]) for VIA; 94.4% (95% CI [84.6, 98.8]) and 73.9% (95% CI [69.7, 78.2]) for HR HPV; and 72.3% (95% CI [59.1, 85.6]) and 93.2% (95% CI [90.8, 95.7]) for cervicography. Conclusion: The pap smear had the highest specificity (94.6%) and HPV testing the highest sensitivity (94.4%). The visual methods, VIA and cervicography, were similar and showed an accuracy in between the former two tests

LAMP for Human African Trypanosomiasis: A Comparative Study of Detection Formats

Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl2 before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl2, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl2 adversely affects reaction sensitivity and was unpopular with several observers

Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs

Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human african trypanosomiasis

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use

Using molecular data for epidemiological inference: assessing the prevalence of Trypanosoma brucei rhodesiense in Tsetse in Serengeti, Tanzania

Background: Measuring the prevalence of transmissible Trypanosoma brucei rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessing human disease risk and monitoring spatio-temporal trends and the impact of control interventions. Although an important epidemiological variable, identifying flies which carry transmissible infections is difficult, with challenges including low prevalence, presence of other trypanosome species in the same fly, and concurrent detection of immature non-transmissible infections. Diagnostic tests to measure the prevalence of T. b. rhodesiense in tsetse are applied and interpreted inconsistently, and discrepancies between studies suggest this value is not consistently estimated even to within an order of magnitude. Methodology/Principal Findings: Three approaches were used to estimate the prevalence of transmissible Trypanosoma brucei s.l. and T. b. rhodesiense in Glossina swynnertoni and G. pallidipes in Serengeti National Park, Tanzania: (i) dissection/microscopy; (ii) PCR on infected tsetse midguts; and (iii) inference from a mathematical model. Using dissection/microscopy the prevalence of transmissible T. brucei s.l. was 0% (95% CI 0–0.085) for G. swynnertoni and 0% (0–0.18) G. pallidipes; using PCR the prevalence of transmissible T. b. rhodesiense was 0.010% (0–0.054) and 0.0089% (0–0.059) respectively, and by model inference 0.0064% and 0.00085% respectively. Conclusions/Significance: The zero prevalence result by dissection/microscopy (likely really greater than zero given the results of other approaches) is not unusual by this technique, often ascribed to poor sensitivity. The application of additional techniques confirmed the very low prevalence of T. brucei suggesting the zero prevalence result was attributable to insufficient sample size (despite examination of 6000 tsetse). Given the prohibitively high sample sizes required to obtain meaningful results by dissection/microscopy, PCR-based approaches offer the current best option for assessing trypanosome prevalence in tsetse but inconsistencies in relating PCR results to transmissibility highlight the need for a consensus approach to generate meaningful and comparable data

Diagnostic Accuracy of Molecular Amplification Tests for Human African Trypanosomiasis—Systematic Review

A range of molecular amplification techniques has been developed for the diagnosis of HAT, with polymerase chain reaction (PCR) at the forefront. As laboratory strengthening in endemic areas increases, it is expected that the applicability of molecular tests will increase. However, careful evaluation of these tests against the current reference standard, microscopy, must precede implementation. Therefore, we have investigated the published diagnostic accuracy of molecular amplification tests for HAT compared to microscopy for both initial diagnosis as well as for disease staging